KR20220132454A - Vaccine composition based on attenuated reovirus and Use thereof - Google Patents

Abstract

The present invention relates to a vaccine composition based on an attenuated reovirus and a use thereof. The attenuated reovirus according to the present invention is obtained by cutting amino acids of 251 - 455 in a sigma 1 protein of a capsid, wherein when an epitope of an antigen protein causing a cancer or an infectious disease is introduced at the cut sigma 1 protein, the epitope of the antigen protein is stably expressed within a cell to cause an immune response, such as generate a neutralizing antibody or induce a cell-mediated immunity. Therefore, when the epitope of the antigen protein is introduced at the cut sigma 1 protein of the attenuated reovirus, it is expected to be useful as a vaccine composition for a cancer and an infectious disease.

Description

Vaccine composition based on attenuated reovirus and Use thereof

The present invention relates to attenuated reovirus based vaccine compositions and uses thereof.

Viruses are small, non-cellular organisms made up of genetic material and proteins. There are different types of viruses. For example, the virus may be a DNA virus that replicates in the nucleus of a host, or an RNA virus that replicates in the cytoplasm of a cell. Viruses may be double-stranded or single-stranded. Furthermore, single-stranded RNA viruses can be either the positive (+, sense) strand or the negative (-) strand. These different types of viruses cause a variety of viral infections.

Viral infection occurs when a pathogenic virus penetrates and enters the body of an organism. Once a virus enters the body of an organism, it attaches itself to cells and reprograms the cells to replicate the new virus until the cell bursts or dies Viruses cause a variety of infectious diseases and allow them to spread rapidly. Infectious diseases caused by viruses usually include colds, flu and warts. However, the virus also affects AIDS, coronavirus infection-19 (COVID-19), hepatitis C, tuberculosis, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), smallpox, herpes. ), hemorrhagic fever, polio, measles, mumps, rubella, rotavirus, and norovirus.

Vaccination is the most important protective measure against viral infection and limiting its spread today. Modern vaccines, as a rule, induce the formation of antibodies to surface viral antigens. Vaccine effectiveness is directly dependent on the degree of agreement between the antigenic structure of the virus contained in the vaccine and the strain circulating in the population. The surface proteins of most viruses undergo constant antigenic variation (antigenic drift), requiring continuous renewal of the vaccine strain composition. The development of highly immunogenic and safe vaccines that induce a broad-acting immune response is currently one of the major problems encountered in effective cancer or infectious disease prevention. The development of a vaccine is necessary to prevent the spread and continuous infection of global epidemic diseases such as coronavirus infection-19 (COVID-19) caused by SARS-CoV-2, which has recently spread. Accordingly, many virus-based vaccines are currently being developed worldwide using platforms such as vesicular stomatitis virus (VSV), measles virus (MeV), adenovirus (Ad), baculovirus, and the like.

Respiratory Enteric Orphan Virus (Reovirus) is a non-enveloped virus whose entire genome consists of 10 double-stranded RNA fragments, is ubiquitous in the general environment, and does not show symptoms in hosts with normal immune function. It is an asymptomatic virus. It is known that the site of infection is limited to the upper respiratory tract and gastrointestinal tract due to its low infectivity to humans.

On the other hand, reverse genetics technology creates a recombined artificial virus by confirming the entire genome sequence of the virus. that there is Various mechanisms such as chimeric virus, codon pair deoptimization, and mutagenesis can be loaded into the virus's gene to create a new engineered virus. The classical genetics approach is mainly a method of randomly inducing mutations in the genome of a given organism and then excavating genes from mutations representing the trait of interest. Mutations in genes have the disadvantage that it is not easy to isolate them. Recent developments in functional genomes have made these mutations available at the genomic level in several model organisms, including yeast. On the other hand, reverse genetics technology can increase the discrimination ability of genetic search by directly introducing and searching possible mutations that can be caused by mutation of each gene using the mutation among several mechanisms. The reverse genetics technique is a very useful technique in vaccine development because it has the advantage of being able to artificially manipulate a virus as desired and maximizing its characteristics.

Therefore, the present inventors use reverse genetics technology to insert an epitope nucleotide sequence so that a specific amino acid sequence position is cut and an epitope for cancer or an infectious disease is fused to the attenuated reovirus sigma 1 protein for expression "quick exchange" Through this method, it was attempted to develop an attenuated reovirus-based vaccine platform.

Korean Patent Publication No. 10-2019-0092468

The present inventors introduced the amino acid sequence of various epitopes for cancer or infectious disease at the cleaved amino acid position in the attenuated reovirus sigma 1 protein by cleaving amino acids 251-455 by introducing a method of substitution or insertion to reduce the expression thereof. As a result of the confirmation, it was confirmed that the introduced epitope was stably expressed in cells and exhibited an immune response such as generating a neutralizing antibody or inducing cellular immunity. Based on this, the present invention was completed.

Accordingly, an object of the present invention is the amino acid sequence of the attenuated reovirus sigma 1 protein; And to provide a polypeptide comprising an amino acid sequence of an epitope causing cancer or an infectious disease, and a polynucleotide encoding the same.

Another object of the present invention is to provide an attenuated reovirus-based viral vector comprising a polynucleotide encoding the polypeptide.

Another object of the present invention is to provide an attenuated reovirus-based vaccine composition comprising the polypeptide, a polynucleotide encoding the same, or the viral vector effectively, and a method for preparing the same.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned may be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.

In order to achieve the above object, the present invention provides an amino acid sequence of an attenuated reovirus sigma 1 protein; And in a polypeptide comprising an epitope amino acid sequence of an antigen causing cancer or an infectious disease,

amino acid sequence 1 to 250 of the reovirus sigma 1 protein attenuated by truncating at amino acid position 251 at the N-terminus; and

The attenuated reovirus sigma 1 protein is inserted into the amino acid position after 251 at the N-terminus of the amino acid sequence of the protein, characterized in that it contains the amino acid sequence of the epitope of an antigen causing cancer or infectious disease, characterized in that it comprises a polypeptide provides

In addition, the present invention provides an attenuated reovirus-based viral vector comprising a polynucleotide encoding the polypeptide.

In addition, the present invention provides an attenuated reovirus-based vaccine composition comprising the polypeptide, a polynucleotide encoding the same, or the viral vector effectively.

The present invention also provides a method for preparing an attenuated reovirus-based vaccine composition comprising the steps of:

a) preparing a reverse-evolved viral vector to include a nucleotide sequence encoding the attenuated reovirus sigma 1 protein represented by SEQ ID NO: 1; and

b) At the 763 to 1416 nucleotide sequence positions of the nucleotide sequence encoding the sigma 1 protein, the nucleotide is substituted or inserted so as to include a nucleotide sequence encoding an epitope of an antigen causing cancer or infectious disease to be attenuated Introducing a polynucleotide encoding the reovirus sigma 1 protein and an epitope of an antigen causing cancer or an infectious disease to be expressed by fusion into the vector.

In one embodiment of the present invention, the attenuated reovirus sigma 1 protein may be represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In another embodiment of the present invention, the attenuated reovirus sigma 1 protein may be encoded by the nucleotide sequence shown in SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the epitope may form or constitute a fusion protein at the carboxy terminus of the attenuated reovirus sigma 1 protein, but is not limited thereto.

As another embodiment of the present invention, one selected from the group consisting of a linker (SEQ ID NO: 11), Myc protein (SEQ ID NO: 12), FLAG protein (SEQ ID NO: 13), and 2A peptide before and after the amino acid sequence of the epitope It may further include the above amino acid sequence, but is not limited thereto.

In another embodiment of the present invention, the cancer is liver cancer, glioma, sarcoma, colorectal cancer, breast cancer, prostate cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, stomach cancer, esophageal cancer, bile duct cancer, pancreatic cancer, cervical cancer , skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, kidney cancer, rectal cancer, and may be one or more selected from the group consisting of brain tumor, but is not limited thereto.

In another embodiment of the present invention, the infectious disease is coronavirus infection-19 (COVID-19), hepatitis C, influenza, human immunodeficiency virus (HIV) induced AIDS (AIDS), tuberculosis, severe acute respiratory syndrome ( SARS), Middle East Respiratory Syndrome (MERS), infantile enteritis caused by rotavirus, and non-bacterial acute gastroenteritis caused by norovirus may be at least one selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of the epitope may be one or more selected from the group consisting of SEQ ID NOs: 3 to 10, but is not limited thereto.

In another embodiment of the present invention, the nucleotide sequence encoding the epitope may be substituted or inserted at positions 763 to 1416 nucleotides from the 5' end of the nucleotide sequence encoding the attenuated reovirus sigma 1 protein. , but not limited thereto.

In another embodiment of the present invention, the epitope may generate an immune response by generating a neutralizing antibody or inducing cellular immunity in the host, but is not limited thereto.

In another embodiment of the present invention, the epitope may be one or more selected from the group consisting of CD4+ T cells, CD8+ T cells, and B-cell epitopes, but is not limited thereto.

In another embodiment of the present invention, the vaccine composition may be for the prevention or treatment of cancer or infectious disease, but is not limited thereto.

In another embodiment of the present invention, in the method for preparing the vaccine composition, c) the attenuated reovirus-based vector comprising the polynucleotide of step b) is BHK21, L929, HEK293, CHO, PER.C6 , HeLa, and Vero cells to produce and propagate in one or more cells selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a method for preventing or treating cancer or an infectious disease, comprising administering the vaccine composition to an individual in need thereof.

In addition, the present invention provides the use of the vaccine composition for the prevention or treatment of cancer or infectious diseases.

In addition, the present invention provides the use of the polypeptide according to the present invention, a polynucleotide encoding the same, or the viral vector for the preparation of a vaccine for the prevention or treatment of cancer or infectious disease.

In the attenuated reovirus according to the present invention, amino acids 251-455 are cleaved from the sigma 1 protein of the capsid. The antigenic protein epitope is stably expressed in the cell, and has the effect of exhibiting an immune response, such as generating a neutralizing antibody or inducing cellular immunity. Accordingly, by introducing an epitope of an antigenic protein into the cleaved sigma 1 protein region of the attenuated reovirus according to the present invention, it is expected to be usefully used as a vaccine composition for cancer or infectious diseases.

1A is a view showing a comparison of the structures of a wild-type reovirus and an attenuated reovirus RP116 according to an embodiment of the present invention.
1B is a schematic diagram showing that a reovirus vaccine platform can be generated by adding epitopes of various pathogen-derived antigens to the cleaved site of the sigma 1 protein of the attenuated reovirus RP116 according to an embodiment of the present invention; to be.
1C is a diagram schematically illustrating an administration route and action of a vaccine prepared using an attenuated reovirus according to an embodiment of the present invention.
2 is a diagram schematically illustrating a quick exchange reovirus reverse genetics system and a reovirus propagation process according to an embodiment of the present invention.
3A is a wild-type reovirus (WT ReoV), an attenuated reovirus RP116 with truncated σ1 (ReoV+Q251 ^* ) and a reovirus introduced with RBD (ReoV+RBD) into L929 cells according to an embodiment of the present invention. ) is a view confirming the results of protein detection by anti-reovirus antibodies after each infection.
Figure 3b is a view confirming that the RBD gene is detected in the genetic material extracted by infecting ReoV + RBD in L929 cells according to an embodiment of the present invention.
Figure 3c is a SARS-CoV-2 neutralizing antibody (NeuAb) (top) and anti-Reovirus antibody (bottom) in a cell lysate obtained by infecting L929 cells with ReoV + RBD according to an embodiment of the present invention. It is a figure confirming the expression of viral protein and RBD.
Figure 4a is a diagram showing a schematic diagram of the design of the recombinant σ1 protein construction of the attenuated reovirus according to an embodiment of the present invention.
Figure 4b is a western blot showing that various antigens introduced from the recombinant reovirus are expressed by engineering to express various CD4+ and CD8+ T cell epitopes by fusion with σ1 protein in the attenuated reovirus according to an embodiment of the present invention. This is a drawing that has been verified through
Figure 4c is a view confirming the expression of the antigen introduced in the recombinant reovirus used in Figure 4b through RT-qPCR analysis.
Figure 4d shows that various B-cell epitopes are expressed by fusion with σ1 protein in the attenuated reovirus according to an embodiment of the present invention, and various antigens introduced from the recombinant reovirus are expressed through western blot. It is a drawing.
Figure 4e is a view confirming the expression of the antigen introduced in the recombinant reovirus used in Figure 4d through RT-qPCR analysis.
5 is a view confirming epitope expression in ReoV+OVA ^257-264 according to an embodiment of the present invention by immunocytochemical analysis.
6 is a view confirming the expression of a linear B cell epitope of SARS-CoV-2 in ReoV-S21P2(2) according to an embodiment of the present invention.
Figure 7a is a diagram showing a schematic diagram of the design of the recombinant σ1 protein construction of the recombinant reovirus ReoV+S21P2(1) and ReoV+OVA ^257-264 according to an embodiment of the present invention.
7B is a view confirming the stability of the introduced epitope by detecting antigens with a reovirus-specific antibody (upper) and a FLAG tag-specific antibody (lower) in a recombinant reovirus according to an embodiment of the present invention.

In one embodiment of the present invention, the attenuated reovirus RP116 with a unique STOP mutation from nucleotide ⁷⁶³ CAA (amino acid ²⁵¹ Q-glutamine) to ⁷⁶³ TAA (STOP codon) is used, and the cleaved sigma 1 protein of RP116 is an epitope amino acid Sequences were added and an attenuated reovirus-based vaccine platform was constructed via a quick exchange reovirus reverse genetics system (see Examples 1 and 2).

In another embodiment of the present invention, as a proof-of-concept for a reovirus vaccine platform, codon-optimized SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) receptor binding domain (RBD) in the form of a fusion protein with sigma 1 protein of reovirus ) was prepared, and it was confirmed that detection by a SARS-CoV-2 neutralizing antibody was possible, confirming that ReoV expressing RBD produced a neutralizing antibody ideal for a vaccine candidate (implementation). See Example 3).

In another embodiment of the present invention, various CD4+ and CD8+ T cell epitopes were engineered to be expressed by fusion with the attenuated reovirus sigma 1 protein using a fast exchange technique, and it was confirmed through experiments that the epitopes are actually expressed. and (see Examples 4 to 6), it was confirmed that stable expression is possible without mutation in the introduced epitope (see Example 7).

Accordingly, the present invention provides an amino acid sequence of an attenuated reovirus sigma 1 protein; And in a polypeptide comprising an epitope amino acid sequence of an antigen causing cancer or an infectious disease,

amino acid sequence 1 to 250 of the reovirus sigma 1 protein attenuated by truncating at amino acid position 251 at the N-terminus; and

The attenuated reovirus sigma 1 protein is inserted into the amino acid position after 251 at the N-terminus of the amino acid sequence of the protein, characterized in that it contains the amino acid sequence of the epitope of an antigen causing cancer or infectious disease, characterized in that it comprises a polypeptide provides

In the present invention, "reovirus (respiratory enteric orphan virus, REO virus)" is a non-enveloped icosahedral virus having a double-stranded RNA fragment as a genome. Reovirus is commonly isolated from the digestive and respiratory tract of healthy humans and is considered a non-pathogenic virome. Reovirus is known as an oncolytic virus capable of infecting and killing various transformed cells.

In the present invention, the “attenuated reovirus” is mutated from ⁷⁶³ CAA to ⁷⁶³ TAA (termination codon) encoding glutamine (Q), which is the 251 amino acid in the attachment protein sigma 1 exposed in the capsid of the wild-type reovirus. Occurs, and after the 251 amino acid of the sigma 1 protein is cleaved, it may be reovirus RP116 characterized in that the globular head form is cleaved.

In the present invention, the attenuated reovirus sigma 1 protein may be represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, the attenuated reovirus sigma 1 protein may be encoded by the nucleotide sequence shown in SEQ ID NO: 2, but is not limited thereto.

In the present invention, “cancer” refers to an aggressive characteristic in which cells divide and grow ignoring normal growth limits, an invasive characteristic that penetrates into surrounding tissues, and a metastatic characteristic that spreads to other parts of the body. ) is a generic term for diseases caused by cells with characteristics.

In the present invention, the type of cancer is not particularly limited as long as it is known as a malignant tumor in the art, for example, liver cancer, glioma, sarcoma, colorectal cancer, breast cancer, prostate cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer , bladder cancer, stomach cancer, esophageal cancer, bile duct cancer, pancreatic cancer, cervical cancer, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, endometrial cancer, kidney cancer, rectal cancer, and may be one or more selected from the group consisting of brain tumors.

In the present invention, "infection" means that a pathogenic microorganism such as a virus invades the body of a host organism, develops and proliferates, and it means that it settles and proliferates on the tissues, body fluids, and surfaces of humans, animals, and plants, As a result, the host may undergo pathological changes and disease may develop.

In the present invention, "infectious disease" means a disease caused by the growth of pathogenic microorganisms by settling on the tissues, body fluids, and surfaces of humans, animals, and plants, and can be divided into several types depending on the route of infection and whether it is contagious. . Such infections include viral infections, fungal infections, bacterial infections, protozoal infections and parasitic infections. In the present invention, the infectious disease is hepatitis C, influenza, human immunodeficiency virus (HIV) induced AIDS (AIDS), tuberculosis, coronavirus infection-19 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS) -CoV-2) may be one or more selected from the group consisting of infectious diseases, infantile enteritis caused by rotavirus, and non-bacterial acute gastroenteritis caused by norovirus, according to an embodiment of the present invention, coronavirus infection -19, but is not limited thereto.

In the present invention, "coronavirus infection-19 (COVID-19)" is a severe respiratory syndrome caused by SARS-CoV-2. In December 2019, the first case was reported in China, and as it spread worldwide, it became an epidemic. Symptoms of COVID-19 vary, but include fever, cough, headache, fatigue, shortness of breath, and loss of smell and taste. Symptoms appear within 1 to 14 days of being infected with the virus. In particular, one-third of infected people are asymptomatic and do not show any noticeable symptoms. 81% of people conspicuous enough to be classified as patients develop mild to severe symptoms, 14% of people develop symptoms such as shortness of breath and hypoxia, and 5% of people develop severe symptoms such as respiratory failure and shock. Occurs. Older adults are more likely to develop severe symptoms, and organ damage has been observed in some people due to COVID-19 exposure long after recovery.

SARS-CoV-2 is 30 kb nucleotides and has four important structural proteins; Nucleocapsid (N), Spike (S), Membrane (M), Envelope (E) proteins. Among them, the S protein is an important site for binding to the receptor of the host cell, and transfers the viral nucleocaspid into the cell and replication occurs.

The simplest and most direct way to combat SARS-CoV-2 is to neutralize the virus that enters human cells. SARS-CoV-2 enters the cell, where replication occurs, and new virions are secreted to infect other cells. blocking the mechanism.

SARS-CoV-2 is known to be capable of viral replication by binding to the angiotensin converting enzyme 2 (ACE2) receptor in human cells. The receptor-binding domain (RBD) of the S protein of the coronavirus is a key domain that binds to the ACE2 receptor in the host cell, and multiple conformational-dependent epitopes that induce potent neutralizing antibodies against SARS-CoV-2 infection. ) could be a key target for the treatment and vaccine development of COVID-19 (J. Immunol 2005;174:4908-4915).

In addition, the present invention provides an attenuated reovirus-based viral vector comprising a polynucleotide encoding the polypeptide.

In addition, the present invention provides an attenuated reovirus-based vaccine composition comprising the polypeptide, a polynucleotide encoding the same, or the viral vector effectively.

In the present invention, the vaccine composition may be for the prevention or treatment of cancer or infectious disease, but is not limited thereto.

In the present invention, “vaccine” refers to a composition containing at least one immunologically active ingredient that induces an immunological response in an animal. The immunologically active component of the vaccine may contain appropriate elements of live or dead virus (subunit vaccines), whereby these elements destroy the whole virus or its growing culture and then the desired construct(s) by synthetic procedures followed by isolation and purification induced by purification steps to obtain It is prepared by induction of the above synthetic process in animals in need of the vaccine by direct incorporation of genetic material using (polynucleotide vaccination).

A vaccine may comprise one or more than one of the elements described above and may be prepared by methods known in the art. In the present invention, the vaccine is based on a reovirus attenuated by cleavage of the sigma 1 protein, and can be prepared so that epitopes causing various cancers or infectious diseases are expressed in a fused form at the cleaved site. For example, a polypeptide comprising the truncated sigma 1 protein and epitope amino acid sequence of the attenuated reovirus, a polynucleotide encoding the same, and a viral vector comprising the polynucleotide may be prepared in the form of, but limited thereto doesn't happen The vaccine of the present invention may be in any form known in the art, for example, in the form of solutions and injections, or in solid form suitable for suspension, but is not limited thereto. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or may be prepared in the form of an aerosol or spray. They may also be incorporated into transdermal patches.

The vaccine according to the present invention may include, if necessary, a pharmaceutically acceptable vaccine protective agent, immune enhancing agent, diluent, absorption promoter, and the like. The vaccine protection agent includes, for example, a mixture of lactose phosphate glutamate gelatin, but is not limited thereto. When the vaccine is a solution or injection, it may contain propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (eg, about 1%) if necessary.

There is no particular limitation on the substance used as the adjuvant, for example, alum, monophosphoryl lipid A (MPL), aluminum hydroxide, mineral oil or other oils, or added to vaccines, or each induction by these additional components. It may be, but is not limited to, an auxiliary molecule generated by the body later.

In this case, "pharmaceutically acceptable" means that when it is physiologically acceptable and administered to an individual, it does not inhibit the action of the active ingredient and is non-toxic that does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. do.

The vaccine may be administered through an administration route such as oral, transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, but is not limited thereto, and for example, may be administered via oral or intramuscular administration routes.

In the present invention, the term “epitope” is an antigenic determinant, and refers to a specific part of an antigen that allows the immune system, such as an antibody, B cell, T cell, etc. to identify the antigen. In order to elicit a B cell (ie, antibody) response, the antigen must contain a “B cell epitope”, and to elicit a T cell response, the antigen must contain a “T cell epitope”. As is generally understood in the art, a B cell epitope is a portion of an antigen that is recognized and bound by a B cell receptor. Lipids, polysaccharides and proteins/peptides may contain B cell epitopes, which, when introduced into a selected organism, cause the B cells to produce antibodies that specifically bind to the introduced epitope.

In the present invention, the epitope may be at least one selected from the group consisting of CD4+ T cells, CD8+ T cells, and B-cell epitopes, and according to an embodiment of the present invention, RBD (SEQ ID NO: 3), OVA ^257-264 (SEQ ID NO: 4), OVA ^323-339 (SEQ ID NO: 5), Adpgk (SEQ ID NO: 6), Rpl18 (SEQ ID NO: 7), P15E (SEQ ID NO: 8), S21P2(1) (SEQ ID NO: 9), and S21P2 ( 2) (SEQ ID NO: 10) may be an epitope comprising one or more amino acid sequences selected from the group consisting of, but is not limited thereto.

In the present invention, the epitope may form or constitute a fusion protein at the carboxy terminus of the attenuated reovirus sigma 1 protein, but is not limited thereto.

In the present invention, the nucleotide sequence encoding the epitope may be substituted or inserted at positions 763 to 1416 nucleotides from the 5' end of the nucleotide sequence encoding the attenuated reovirus sigma 1 protein, but is not limited thereto. does not For example, the nucleotide sequence encoding the epitope may be substituted for the nucleotide sequence at a specific position among the nucleotide sequence positions 763 to 1416 from the 5' end in the nucleotide sequence of SEQ ID NO: 2 encoding the attenuated reovirus sigma 1 protein. Or, it may be inserted between nucleotide sequences at specific positions. Accordingly, in the amino acid sequence of the wild-type reovirus sigma 1 protein, after the 251 amino acid sequence from the N-terminus is cleaved, and in the attenuated reovirus sigma 1 protein, it is inserted into the amino acid position after the 251st and is fused The epitope to be used may include, but is not limited to, a sequence of about 5 to 400 amino acids.

In the present invention, a linker (SEQ ID NO: 11), Myc protein (SEQ ID NO: 12) before and after the amino acid sequence of the epitope included in the amino acid position after 251 in the sigma 1 protein of the attenuated reovirus , FLAG protein (SEQ ID NO: 13), and may further include one or more amino acid sequences selected from the group consisting of 2A peptides, but is not limited thereto, and the order thereof is not limited. The 2A peptide may be selected from 2A peptides including, for example, P2A, T2A, E2A, or F2A, and the type thereof is not limited.

In the present invention, one or more epitopes selected from the group consisting of CD4+ T cells, CD8+ T cells, and B-cell epitopes allow the T cells or B cells to discriminate antigens, thereby producing neutralizing antibodies or promoting cellular immunity in the host. It may be to cause an immune response by inducing, but is not limited thereto.

In the present invention, "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include numerous immunoglobulin variable region genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains were classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, consequently defining the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody becomes most critical in binding specificity and affinity. Antibodies are major players in humoral immunity, play an important role in living body defense mechanisms together with sensitized lymphocytes, and are produced by administering antigenic proteins to animals as immunogens to induce humoral immune responses. Antibodies or fragments of antibodies may be derived from different individuals, including, but not limited to, humans, mice, rats, hamsters, camels, rabbits, and the like.

In the present invention, "neutralizing antibody" refers to any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the pathogen's ability to infect cells or cause disease.

In the present invention, the term “cellular immunity” refers to an immune activity in which a lymphoid cell directly induces an immune response to an antigen. White blood cells act on an antigen to eat the cell, phagocytosis, or toxic cell reaction to remove the antigen refers to the immune activity. It constitutes the immune system together with the humoral immunity performed by serum antibodies in the body, and a typical example is the action of T cells having cytotoxicity. T cells bind to B cells to form antibodies, and then directly contact antigens to destroy antigens.

In the present invention, "antigen" refers to a protein having the ability to generate an immune response in a host. An antigen can be recognized by and bound to the antibody. The antigen may be derived from the body or the external environment, and also includes a recombinant form of the protein. In the present invention, the vaccine composition can induce the production of an antibody against an epitope of an antigen that causes cancer or an infectious disease, wherein the epitope acts as an antigen. When the vaccine composition is administered twice or three times compared to when the vaccine composition is administered once, antibody production may be further increased, but is not limited thereto.

The present invention also provides a method for preparing an attenuated reovirus-based vaccine composition comprising the steps of:

a) preparing a reverse-evolved viral vector to include a nucleotide sequence encoding the attenuated reovirus sigma 1 protein represented by SEQ ID NO: 1; and

b) At the 763 to 1416 nucleotide sequence positions of the nucleotide sequence encoding the sigma 1 protein, the nucleotide is substituted or inserted so as to include a nucleotide sequence encoding an epitope of an antigen causing cancer or infectious disease to be attenuated Introducing a polynucleotide encoding the reovirus sigma 1 protein and an epitope of an antigen causing cancer or an infectious disease to be expressed by fusion into the vector.

In the present invention, the reverse-evolved viral vector may refer to a viral vector prepared to produce an S1 segment RNA encoding an attenuated reovirus sigma 1 protein in a host cell, and is performed by a method known in the art. can be manufactured. The vector is constructed so that the viral RNA segment can be transcribed from the T7 RNA polymerase promoter, and the 3' end is naturally formed by the ribozyme in the vector. RNA is produced and viral proteins are synthesized using it, which is called a reverse genetics system. This is a useful method for designing and creating RNA virus mutants.

In the method for preparing the vaccine composition of the present invention, c) the attenuated reovirus-based vector comprising the polynucleotide of step b) is transformed into BHK21, L929, HEK293, CHO, PER.C6, HeLa, and Vero cells. It may include the step of producing and proliferating in one or more cells selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a method for preventing or treating cancer or an infectious disease, comprising administering the vaccine composition to an individual in need thereof.

In addition, the present invention provides the use of the vaccine composition for the prevention or treatment of cancer or infectious diseases.

In addition, the present invention provides the use of the polypeptide according to the present invention, a polynucleotide encoding the same, or the viral vector for the preparation of a vaccine for the prevention or treatment of cancer or infectious disease.

In the present invention, “prevention” means any action that suppresses or delays the onset of a desired disease, and “treatment” means that the desired disease and metabolic abnormalities are improved or beneficial by administration of the vaccine composition according to the present invention. Any action that changes.

As used herein, the term “administration” means providing a given vaccine composition of the present invention to a subject by any suitable method.

As used herein, the term “subject” refers to a subject in need of prevention of cancer or infectious disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, and mammals such as cattle.

In the present invention, when the term “comprising” is used, it means that other components may be further included, rather than excluding other components, unless otherwise stated. The term “step of (to)” or “step of” as used throughout the present invention does not mean “step for”.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Construction of an attenuated reovirus-based vaccine platform

Reoviruses are double-stranded RNA non-enveloped viruses. REO stands for Respiratory Enteric Orphan, which has been isolated for the human respiratory and intestinal tract, but is not associated with any known human disease. Most of the neutralizing antibodies and immune responses resulting from wild-type reovirus infection are directed against the viral sigma 1 (σ1) protein, more specifically the globular head of the protein protruding from the capsid. Thus, this region of σ1 for reovirus represents a key part of enhancing antigenicity. As shown in FIG. 1A , wild-type (WT) reovirus recognizes and binds to the junction adhesion molecule-A (JAM-A) and displays the attachment protein σ1 on the entry outer capsid (left side of FIG. 1A).

The amino acid sequence and base sequence of the wild-type reovirus σ1 protein are shown in Table 1 below.

order SEQ ID NO: RP116 sigma-1 protein MDPRLREEVVRLIIALTSDNGVSLSKGLESRVSALEKTSQIHSDTILRITQGLDDANKRIIALEQSRDDLVASVSDAQLAISRLESSIGALQTVVNGLDSSVTQLGARVGQLETGLAELRVDHDNLVARVSLSKGLESRVSALEKTSQIHSDTILRITQGLDDANKRIIALEQSRDDLVASVSDAQLAISRLESSIGALQTVVNGLDSSVTQLGARVGQLETGLAELRVDHDNLVARVSLSKGLESRNIGSLTTELSTLTLRVTSIQFRNIGSLTTELSTLTLRVTSIQADFESGNAIRTLERTAVTSGLDSFNDQSNLAIRTLERTAVTS One RP116 sigma-1 gene GCTATTGGTCGGATGGATCCTCGCCTACGTGAAGAAGTAGTACGGCTGATAATCGCATTAACGAGTGATAATGGAGTATCACTGTCAAAAGGGCTTGAATCAAGGGTCTCGGCGCTCGAGAAGACGTCTCAAATACACTCTGATACTATCCTCCGGATCACCCAGGGACTCGATGATGCAAACAAACGAATCATCGCTCTTGAGCAAAGTCGGGATGACTTGGTTGCATCAGTCAGTGATGCTCAACTTGCAATCTCCAGATTGGAAAGCTCTATCGGAGCCCTCCAAACAGTTGTCAATGGACTTGATTCGAGTGTTACCCAGTTGGGTGCTCGAGTGGGACAACTTGAGACAGGACTTGCAGAGCTACGCGTTGATCACGACAATCTCGTTGCGAGAGTGGATACTGCAGAACGTAACATTGGATCATTGACCACTGAGCTATCAACTCTGACGTTACGAGTAACATCCATACAAGCGGATTTCGAATCTAGGATATCCACATTAGAGCGCACGGCGGTCACTAGCGCGGGAGCTCCCCTCTCAATCCGTAATAACCGTATGACCATGGGATTAAATGATGGACTCAYGTTGTCAGGGAATAATCTCGCCATCCGATTGCCAGGAAATACGGGTCTGAATATTCAAAATGGTGGACTTCAGTTTCGATTTAATACTGATCAATTCCAGATAGTTAATAATAACTTGACTCTCAAGACGACTGTGTTTGATTCTATCAACTCAAGGABAGGCGCAAYTGAGTAAAGTKMCGTGGCGTCGGCAGTGACTCCCTTGAGATTAAACAGTAGCACGAAGGTGCTGGATATGCTAATAGACAGTTCAACACTTGAAATTAATTCTAGTGGACAGCTAACTGTTAGATCGACATCCCCGAATTTGAGGTATCCGATAGCTGATGTTAGCGGCGGTATCGGAATGAGTCCAAATTATAGGTTTAGGCAGAGCATGTGGATAGGAATTGTCTCCTATTCTGGTAGTG GGCTGAATTGGAGGGTACAGGTGAACTCCGACATTTTTATTGTAGATGATTACATACATATATGTCTTCCAGCTTTTGACGGTTTCTCTATAGCTGACGGTGGAGATCTATCGTTGAACTTTGTTACCGGATTGTTACCACCGTTACTTACAGGAGACACTGAGCCCGCTTTTCATAATGACGTGGTCACATATGGAGCACAGACTGTAGCTATAGGGTTGTCGTCGGGTGGTGCGCCTCAGTATATGAGTAAGAATCTGTGGGTGGAGCAGTGGCAGGATGGAGTACTTCGGTTACGTGTTGAGGGGGGTGGCTCAATTACGCACTCAAACAGTAAGTGGCCTGCCATGACCGTTTCGTACCCGCGTAGTTTCACGTGAGGATCAGACCACCCCGCGGCACTGGGGCATTTCATC 2

Attenuated reovirus RP116 is a reovirus with a unique STOP mutation from nucleotide ⁷⁶³ CAA (amino acid ²⁵¹ Q-glutamine) to ⁷⁶³ TAA (STOP codon), which produces a “head truncated” σ1 protein compared to WT reovirus σ1. created (right side view of Fig. 1A).

As shown in Figure 1b, this σ1 protein of RP116 shows that the globular head is not essential for reovirus replication and can be exchanged with other antigenic fragments from various pathogens at this site. About 5 to 400 amino acids of the epitope fragment can be added to this region of the RP116 σ1 protein for the creation of an innovative and safe reovirus vaccine platform.

In addition, as shown in FIG. 1C , the enveloped reovirus is resilient to harsh environmental conditions and can be administered with various solutions or food sources. Upon oral entry, the reovirus preferentially infects the M cells of the small intestine and induces immune responses against the various pathogen epitopes indicated by σ1.

Example 2. Construction of a quick exchange reovirus reverse genetics system

A reverse genetics system for reoviruses was transfected with 10 viral gene segments and a T7-expressing cell line to generate replicable reovirus particles (Kobayshi et al., 2007, Cell Host Microbe. 2007 Apr 19;1(2): 147-57.), then an improved version of the reverse genetics system was reported (Kobayashi et al., 2010, Virology. 2010) requiring only 4 plasmids containing 10 viral gene segments to be transfected into a T7-expressing cell line (Kobayashi et al., 2010, Virology. 2010). Mar 15;398(2):194-200.). In the present invention, a rapid "quick exchange" approach for vaccine design and testing was implemented by constructing a novel reverse genetics system using the attenuated reovirus RP116 gene. The vectors used to introduce the system in the present invention to design a reovirus RP116-based vaccine are as follows (see the upper drawing of FIG. 2): pS1Att or pS1XX, pL1, pSet2, pSet3, pSet4.

When the vector is introduced into BHK21 cells transformed to express T7 RNA polymerase, the viral gene is transcribed and ultimately induces the production of replicable viral particles within 24-72 hours. pS1XX was prepared by introducing a non-reovirus epitope by redesigning the sequence from a STOP codon mutation at amino acid position 251 of pS1Att, ie, the S1 gene of RP116, to the C-terminus. The antigen or epitope fragment can be "fused" to the RP116 σ1 protein sequence by redesigning the sequence after the STOP codon and nucleotide 763. A specific restriction enzyme recognition site was also designed between amino acid position 251 and the C-terminus, so that this part of the σ1 peptide could be easily exchanged.

The resulting replicable particles further facilitated attachment and entry into production cell lines BHK21, L929, Vero cells by dissolving the outer capsid through 50 μg/mL treatment with chymotrypsin prior to infection.

The quick exchange reovirus reverse genetics system and the reovirus propagation process are schematically shown in FIG. 2 .

In addition, the amino acid sequence of the epitope used in the present invention, the construct amino acid sequence, and the codon-optimized construct DNA sequence are shown in Table 2 below.

Epitope Name Amino Acid Sequence Construct Amino Acid Sequence Codon Optimized Construct DNA Sequence (5'-3') RBD NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTR
(SEQ ID NO: 3) NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTR
(SEQ ID NO: 3)
(SEQ ID NO: 21) OVA ^257-264 SIINFEKL
(SEQ ID NO: 4) GGGGSGGGGSEQKLISEEDLSIINFEKLGGGGSSIINFEKLGGGGSSIINFEKLDYKDDDDK
(SEQ ID NO: 14) ggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgAGTATTATAAACTTCGAGAAGCTGGGTGGGGGAGGAAGTTCAATCATTAATTTTGAAAAACTTGGAGGAGGTGGATCTTCCATAATTAATTTTGAAAAGCTGgattataaggatgacgacga
(SEQ ID NO: 22) OVA ^323-339 ISQAVHAAHAEINEAGR
(SEQ ID NO: 5) GGGGSGGGGSEQKLISEEDLISQAVHAAHAEINEAGRDYKDDDDK
(SEQ ID NO: 15) ggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgatttcacaggctgtgcatgctgcacatgctgaaattaatgaggctggacgtgattataaggatgacgacgataagtga
(SEQ ID NO: 23) adpgk ASMTNMELM
(SEQ ID NO: 6) GGGGSGGGGSEQKLISEEDLASMTNMELMGGGGSASMTNMELMGGGGSASMTNMELMDYKDDDDK
(SEQ ID NO: 16) ggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctggctagcatgacgaacatggagctaatgggagggggcggaagtgcttctatgactaatatggaactgatgggaggtgacgactttgcttcaatgacctggattataaggaactta
(SEQ ID NO: 24) Rpl18 KILTFDRL (SEQ ID NO: 7) GGGGSGGGGSEQKLISEEDLKILTFDRLGGGGSKILTFDRLGGGGSKILTFDRLDYKDDDDK
(SEQ ID NO: 17) ggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgaagattctgacgtttgatcgtctggggggggtggcggatctaagatattgacgttcgatcgactgggaggaggtggctctaagatcctaacattcgaccgtctagagattataagga
(SEQ ID NO: 25) P15E KSPWFTTL (SEQ ID NO: 8) GGGGSGGGGSEQKLISEEDLKSPWFTTLGGGGSKSPWFTTLGGGGSKSPWFTTLDYKDDDDK
(SEQ ID NO: 18) gggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgAAGTCACCTTGGTTTACTCTCGGTGGGGGAGGAAGTAAATCGCCATGGTTTACTACATTGGGAGGAGGTGGGTCTAAGTCACCATGGTTCACGACATTGgattataaggatgacgacga
(SEQ ID NO: 26) S21P2(1) PSKPSKRSFIEDLLFNKV
(SEQ ID NO: 9) GGGGSGGGGSEQKLISEEDLPSKPSKRSFIEDLLFNKVDYKDDDDK
(SEQ ID NO: 19) ggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgCCTAGTAAGCCTTCTAAGCGCAGCTTCATAGAAGATTTGTTGTTCAATAAGGTAgattataaggatgacgacgataagtga
(SEQ ID NO: 27) S21P2(2) PSKPSKRSFIEDLLFNKV
(SEQ ID NO: 10) GGGGSGGGGSEQKLISEEDLPSKPSKRSFIEDLLFNKVGGGGSPSKPSKRSFIEDLLFNKVDYKDDDDK
(SEQ ID NO: 20) gggaggtggaggctcaggtggcggaggttctgagcagaagttgatttcagaggaagatctgCCTAGTAAGCCTTCTAAGCGCAGCTTCATAGAAGATTTGTTGTTCAATAAGGTAGGTGGGGGAGGAAGTCCATCGAAGCCAAGTAAGCGTAGTTTCATTGAGGACCTCCTCTCTTCAACAAGGt
(SEQ ID NO: 28)

Example 3. Preparation of ReoV-RBD

As a proof-of-concept for a reovirus vaccine platform, attenuated to express a codon-optimized SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) receptor binding domain (RBD) sequence in the form of a fusion protein with the σ1 protein of reovirus. Recombinant reovirus ReoV+RBD (ReoV-RBD), WT σ1 (WT ReoV) and truncated σ1 prepared by introducing the SARS-CoV-2 receptor binding domain (RBD) base sequence after codon position 251 of the reovirus S1 gene A reovirus with (ReoV+Q251 ^* ) was generated in BHK21 cells and propagated in L929. In this process, a recombinant reovirus expressing a SARS-CoV-2 receptor binding domain (RBD) at σ1 can be detected by a SARS-CoV-2 neutralizing antibody.

As shown in Fig. 3a, L929 cells were infected with wild-type reovirus (WT ReoV), reovirus with truncated σ1 (ReoV+Q251 ^* ) and reovirus introduced with RBD antigen group (ReoV+RBD) for 96 hours, respectively. It was confirmed by Western blot results that each reovirus protein was detected by the anti-reovirus antibody in the cell lysate.

As a result of RT-qPCR analysis after 4 passages of the recombinant reovirus using RBD gene-specific primers from the genetic material extracted by infecting L929 cells with each recombinant reovirus, as shown in FIG. 3b, ReoV+RBD It was confirmed that the RBD gene was detected when infected. Primer sequences used for RT-qPCR analysis are shown in Table 3 below.

Gene Primer 5' - 3' SEQ ID NO: S1
3b, 4c, 4e S1 fwd CACCCAGGGACTCGATGATG 29 S1 rev GCACCCAACTGGGTAACACT 30 RBD
Figure 3b RBD fwd CAACTGAGCAAAGTTACGTGAGGGTACAACCTACGGAATC 31 RBD rev ATGAAATGCCCCAGTGCCGCGGGGTGGTCTGACCTCAAAAATTGACGCATTTATTCTTCAC 32 Epitope
4c, 4e S1-250-AvrII-F GATTCTATCAACTCAAGGATAGGCGCAATTGAGCCTAGG 33 pBacT7-SacII-R CAGTGCCGCGGGGTGGTCTGATCCTCACGTGAAACTACGCGGGTA 34

In addition, as a result of performing a dot blot method on the cell lysate obtained by infecting L929 cells, as shown in FIG. 3C , a SARS-CoV-2 neutralizing antibody (NeuAb) (top) and an anti-reovirus antibody ( It was confirmed that the recombined RBD was expressed by (bottom). The positive control recombinant RBD was prepared by Dr. Manufactured by John Bell laboratory (Azad et al., Membranes (Basel). 2020 Aug 30;10(9):215), SARS-CoV-2 NeuAb (40592-MM57) was Sino Biological (Wayne, PA, USA). was purchased and used. These results indicate that ReoV expressing RBD produces neutralizing antibodies that are ideal for vaccine candidates.

Example 4. Foreign antigen expression using reovirus adhesion protein sigma 1 (σ1)

Specific mutagenic changes can be introduced by site-directed mutagenesis or replication to examine the role or consequences of a defined mutation within one or several reovirus gene segments to cause the reovirus to be altered or mutated. For example, a specific amino acid mutation in the S4 gene of a reovirus encoding a sigma 3 protein is introduced into a viral vector to generate a modified reovirus with a specific mutation in the sigma 3 protein of the viral capsid.

The main mutation of the attenuated reovirus RP116 of the present invention is a STOP mutation from ⁷⁶³ CAA (amino acid ²⁵¹ Q-glutamine) to ⁷⁶³ TAA (STOP codon) in the S1 gene encoding the σ1 protein, and the Production produces a replicable RP116 reovirus with a truncated σ1 protein.

Depending on the characteristics of the gene fragment, substitution or insertion of a specific epitope sequence selected at nucleotide positions 763-1416 is possible, and the substitution or insertion of this non-reovirus sequence essentially replacing the globular head structure of σ1 is the non-reovirus. A novel epitope that generates an immune response to the antigen is exposed to the capsid of the virus.

Figure 4a shows a schematic diagram of the construction of the recombinant attachment protein, and the residue between amino acid position 251 and amino acid position 455 of the σ1 protein can be replaced with various epitopes, including Myc and FLAG tags for labeling purposes.

Reoviruses were engineered to express various CD4+ and CD8+ T cell epitopes by fusion with the σ1 protein using “Quick Exchange” technique, and several types introduced from recombinant reoviruses by western blot as shown in Figure 4b It was confirmed that the antigen was expressed, and the expression of the antigen introduced from the recombinant reovirus used in FIG. 4b was confirmed by separating with a DNA agarose gel by performing RT-qPCR analysis using a gene-specific primer as shown in FIG. 4c. .

In addition, the reovirus was engineered to express various B-cell epitopes by fusion with the σ1 protein using a fast exchange technique, and it was confirmed that various antigens introduced from the recombinant reovirus were expressed by Western blot as shown in Fig. 4d. , It was confirmed by separating with a DNA agarose gel that the antigen introduced from the recombinant reovirus used in FIG. 4d is expressed by performing RT-qPCR analysis using a gene-specific primer as shown in FIG. 4e.

These examples show the versatility of the novel reovirus vaccine platform expressing various foreign antigen epitopes, suggesting that it can be used from the screening stage in the development of new vaccines by introducing various disease epitopes.

Example 5. Confirmation of antigen expression introduced in cells infected with ReoV-OVA

Myc and FLAG tags flanking the OVA ^257-264 epitope in ReoV+ovalbumin (OVA) ^257-264 constructed as shown in FIG. 4A can be detected by immunocytochemical analysis.

To detect this, Vero cells grown on coverslips were infected with ReoV, ReoV+Q251 ^* , and ReoV+OVA ^257-264 for 48 hours. Then, the cells were fixed in 3.7% PFA and reacted with anti-ReoV (1:1000, rabbit serum), anti-Myc (1:200), and anti-FLAG (1:200) for 1 hour, and then anti- -ReoV antibody secondary antibody rabbit Alexa Fluor 488 and anti-Myc or anti-FLAG secondary antibody Alexa Fluor 594 secondary antibody was incubated for 45 minutes. Stained coverslips were mounted with a Fluoromount G with DAPI for nuclear counter staining, and images were taken with an Olympus confocal microscope with a 60X oil objective (scale bar = 50 μm).

As a result, as shown in FIG. 5 , when infected with ReoV+OVA ^257-264 , fluorescence (red) displayed in Myc and FLAG tags overlapped with fluorescence (green) detected for Reovirus protein.

This indicates that the recombinant reovirus specifically expresses antigen in infected cells, suggesting that the recombinant reovirus is infectious and capable of expressing engineered neoepitopes in mammalian cells.

Example 6. Expression and detection of SARS-CoV-2 linear B cell epitope

BHK21 cells were infected with wild-type (WT) or engineered ReoV (ReoV-S21P2(2) expressing ReoV+p15E and SARS-CoV-2 linear B cell epitopes). In a previous study, the SARS-CoV-2 linear B cell epitope was validated to generate neutralizing antibodies (Poh et al., Nat Commun. 2020 Jun 1;11(1):2806).

To confirm the expression of the SARS-CoV-2 linear B cell epitope, cells were lysed 48 hours after infection and used for Western blot. As a result, as shown in FIG. 6 , reovirus protein could be detected in all infected samples, but both Myc and FLAG tags were detected only in ReoV engineered with p15E or SARS-CoV-2 B cell epitopes. In addition, after labeling with an anti-S2 antibody generated from the entire S2 portion of the SARS-CoV-2 spike protein (Sino Biological) containing the B cell epitope region of SARS-CoV-2, ReoV+S21P2(2) infection Only the samples could clearly identify immunoreactive protein bands (indicated by arrows).

These results suggest that a recombinant reovirus harboring a linear B cell epitope of SARS-CoV-2 can induce a specific immune response to the epitope.

Example 7. Confirmation of stability of recombinant reovirus

Figure 7a shows a schematic diagram of the design of the recombinant σ1 protein construction of the recombinant reoviruses ReoV+S21P2(1) and ReoV+OVA ^257-264 .

Each epitope was designed to include Myc and FLAG tags at the N-terminus and C-terminus, respectively, and was used to confirm whether the introduced epitope was stably expressed.

BHK21 cells were infected with recombinant reovirus for 48 hours and analyzed by western blotting, and both reovirus-specific antibodies (top) and FLAG-tag-specific antibodies (bottom) were detected in recombinant reoviruses. A GAPDH recognition antibody (anti-GAPDH) was used as a loading control.

As a result, as shown in FIG. 7b , it was confirmed that a high concentration of antigen was detected with the FLAG tag, and it was found that the detected high concentration antigen can be stably expressed without mutation in the introduced epitope even after 5 passages.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Virocure Inc. <120> Vaccine composition based on attenuated reovirus and Use thereof <130> MP21-052P1 <150> KR 10-2021-0037160 <151> 2021-03-23 <160> 34 <170> KoPatentIn 3.0 <210> 1 <211 > 250 <212> PRT <213> Artificial Sequence <220> <223> RP116 sigma-1 protein <400> 1 Met Asp Pro Arg Leu Arg Glu Glu Val Val Arg Leu Ile Ile Ala Leu 1 5 10 15 Thr Ser Asp Asn Gly Val Ser Leu Ser Lys Gly Leu Glu Ser Arg Val 20 25 30 Ser Ala Leu Glu Lys Thr Ser Gln Ile His Ser Asp Thr Ile Leu Arg 35 40 45 Ile Thr Gln Gly Leu Asp Asp Ala Asn Lys Arg Ile Ile Ala Leu Glu 50 55 60 Gln Ser Arg Asp Asp Leu Val Ala Ser Val Ser Asp Ala Gln Leu Ala 65 70 75 80 Ile Ser Arg Leu Glu Ser Ser Ile Gly Ala Leu Gln Thr Val Val Asn 85 90 95 Gly Leu Asp Ser Ser Val Thr Gln Leu Gly Ala Arg Val Gly Gln Leu 100 105 110 Glu Thr Gly Leu Ala Glu Leu Arg Val Asp His Asp Asn Leu Val Ala 115 120 125 Arg Val Asp Thr Ala Glu Arg Asn Ile Gly Ser Leu Thr Thr Glu Leu 130 135 140 Ser Thr Leu Thr Leu Arg Val Thr Ser Ile Gln Ala Asp Phe Glu Ser 145 150 155 160 Arg Ile Ser Thr Leu Glu Arg Thr Ala Val Thr Ser Ala Gly Ala Pro 165 170 175 Leu Ser Ile Arg Asn Asn Arg Met Thr Met Gly Leu Asn Asp Gly Leu 180 185 190 Xaa Leu Ser Gly Asn Asn Leu Ala Ile Arg Leu Pro Gly Asn Thr Gly 195 200 205 Leu Asn Ile Gln Asn Gly Gly Leu Gln Phe Arg Phe Asn Thr Asp Gln 210 215 220 Phe Gln Ile Val Asn Asn Asn Leu Thr Leu Lys Thr Thr Val Phe Asp 225 230 235 240 Ser Ile Asn Ser Arg Xaa Gly Ala Xaa Glu 245 250 <210> 2 <211> 1416 <212> DNA <213> Artificial Sequence <220> <223> RP116 sigma-1 gene <400> 2 gctattggtc ggatggatcc tcgcctacgt gaagaagtag tacggctgat aatcgcatta 60 acgagtgata atggagtatc actgtcaaaa gggcttgaat caagg gtctc ggcgctcgag 120 aagacgtctc aaatacactc tgatactatc ctccggatca cccagggact cgatgatgca 180 aacaaacgaa tcatcgctct tgagcaaagt cgggatgact tggttgcatc agtcagtgat 240 gctcaacttg caatctccag attggaaagc tctatcggag ccctccaaac agttgtcaat 300 ggacttgatt cgagtgttac ccagttgggt gctcgagtgg gacaacttga gacaggactt 360 gcagagctac gcgttgatca cgacaatctc gttgcgagag tggatactgc agaacgtaac 420 attggatcat tgaccactga gctatcaact ctgacgttac gagtaacatc catacaagcg 480 gatttcgaat ctaggatatc cacattagag cgcacggcgg tcactagcgc gggagctccc 540 ctctcaatcc gtaataaccg tatgaccatg ggattaaatg atggactcay gttgtcaggg 600 aataatctcg ccatccgatt gccaggaaat acgggtctga atattcaaaa tggtggactt 660 cagtttcgat ttaatactga tcaattccag atagttaata ataacttgac tctcaagacg 720 actgtgtttg attctatcaa ctcaaggaba ggcgcaaytg agtaaagtkm cgtggcgtcg 780 gcagtgactc ccttgagatt aaacagtagc acgaaggtgc tggatatgct aatagacagt 840 tcaacacttg aaattaattc tagtggacag ctaactgtta gatcgacatc cccgaatttg 900 aggtatccga tagctgatgt tagcggcggt atcggaatga gtccaaatta taggtttagg 960 cagagcatgt ggataggaat tgtctcctat tctggtagtg ggctgaattg gagggtacag 1020 gtgaactccg acatttttat tgtagatgat tacatacata tatgtcttcc agcttttgac 1080 ggtttctcta tagctgacgg tggagatcta tcgttgaact ttgttaccgg attgttacca 1140 ccgttactta caggagacac tgagcccgct tttcataatg acgtggtcac atatggagca 1200 cagactgtag ctatagggtt gtcgtcgggt ggtgcgcctc agtatatgag taagaatctg 1260 tgggtggagc agtggcagga tggagtactt cggttacgtg ttgagggggg tggctcaatt 1320 acgcactcaa acagtaagtg gcctgccatg accgtttcgt acccgcgtag tttcacgtga 1380 ggatcagacc accccgcggc actggggcat ttcatc 1416 <210> 3 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> RBD Amino Acid Sequence <400> 3 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 1 5 10 15 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 20 25 30 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 35 40 45 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 50 55 60 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 65 70 75 80 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 145 150 155 160 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190 Thr Val <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> OVA257-264 Amino Acid Sequence <400> 4 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 <210> 5 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> OVA323-33 9 Amino Acid Sequence <400> 5 Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly 1 5 10 15 Arg <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> < 223> Adpgk Amino Acid Sequence <400> 6 Ala Ser Met Thr Asn Met Glu Leu Met 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Rpl18 Amino Acid Sequence <400 > 7 Lys Ile Leu Thr Phe Asp Arg Leu 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> P15E Amino Acid Sequence <400> 8 Lys Ser Pro Trp Phe Thr Thr Leu 1 5 <210> 9 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> S21P2(1) Amino Acid Sequence <400> 9 Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn 1 5 10 15 Lys Val <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> S21P2(2) Amino Acid Sequence <400> 10 Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn 1 5 10 15 Lys Val <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 11 Gly Gly Gly Gly Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> c-Myc tag <400> 12 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> FLAG tag <400> 13 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 14 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> OVA257-264 Construct Amino Acid Sequence <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Ser Ile Ile Asn Phe Glu Lys Leu Gly Gly Gly Gly 20 25 30 Ser Ser Ile Ile Asn Phe Glu Lys Leu Gly Gly Gly Gly Ser Ser Ile 35 40 45 Ile Asn Phe Glu Lys Leu Asp Tyr Lys Asp Asp Asp Asp Lys 50 55 60 <210> 15 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> OVA323-339 Construct Amino Acid Sequence <400> 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile 20 25 30 Asn Glu Ala Gly Arg Asp Tyr Lys Asp Asp Asp Asp Lys 35 40 45 <210> 16 <211> 65 <212> PRT <213> Artificial Sequence <220 > <223> Adpgk Construct Amino Acid Sequence <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Ala Ser Met Thr Asn Met Glu Leu Met Gly Gly Gly 20 25 30 Gly Ser Ala Ser Met Thr Asn Met Glu Leu Met Gly Gly Gly Gly Ser 35 40 45 Ala Ser Met Thr Asn Met Glu Leu Met Asp Tyr Lys Asp Asp Asp Asp Asp Asp 50 55 60 Lys 65 <210> 17 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Rpl18 Construct Amino Acid Sequence <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Lys Ile Leu Thr Phe Asp Arg Leu Gly Gly Gly Gly 20 25 30 Ser Lys Ile Leu Thr Phe Asp Arg Leu Gly Gly Gly Gly Ser Lys Ile 35 40 45 Leu Thr Phe Asp Arg Leu Asp Tyr Lys Asp Asp Asp Asp Lys 50 55 60 < 210> 18 <211> 62 <212> PRT <213> Artificial Se sequence <220> <223> P15E Construct Amino Acid Sequence <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Lys Ser Pro Trp Phe Thr Thr Leu Gly Gly Gly Gly 20 25 30 Ser Lys Ser Pro Trp Phe Thr Thr Leu Gly Gly Gly Gly Ser Lys Ser 35 40 45 Pro Trp Phe Thr Thr Leu Asp Tyr Lys Asp Asp Asp Asp Lys 50 55 60 <210> 19 <211> 46 < 212> PRT <213> Artificial Sequence <220> <223> S21P2(1) Construct Amino Acid Sequence <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp 20 25 30 Leu Leu Phe Asn Lys Val Asp Tyr Lys Asp Asp Asp Asp Lys 35 40 45 <210> 20 <211> 69 <212> PRT <213> Artificial Sequence < 220> <223> S21P2(2) Construct Amino Acid Sequence <400> 20 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp 20 25 30 Leu Leu Phe Asn Lys Val Gly Gly Gly Gly Ser Pro Ser Lys Pro Ser 35 40 45 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Asp Tyr Lys 50 55 60 Asp Asp Asp Asp Lys 65 <210> 21 <211> 636 <212> DNA <213> Artificial Sequence <220> <223> RBD Codon Optimized Construct DNA Sequence <400> 21 aatatcacta acttgtgtcc gttcggcgag gtttttaatg cgaccaggtt tgcttccgtg 60 tacgcctgga acaggaaacg gatctccaat tgtgtcgccg attactccgt cttgtataat 120 tcagcatctt tcagcacgtt taaatgttac ggagtttccc ccacaaaatt gaatgacctt 180 tgctttacga acgtctacgc ggattcattt gtaatccggg gggacgaagt taggcaaatt 240 gcgccagggc agactggcaa gatagctgac tataattata aattgccgga tgactttacg 300 ggctgtgtga ttgcttggaa ctcaaataat ctggactcaa aggtaggggg aaattataac 360 tacctttaca ggctgttccg gaagagtaat ctgaagccat tcgaaagaga tataagtaca 420 gagatctacc aagctggaag caccccctgc aatggtgttg aaggattcaa ttgttatttc 480 ccattgcaat cctatggttt tcaaccgacg aatggggtgg gataccaacc atatcgagtt 540 gtggttctca gtttcgagtt gcttcatgct cctgcgacag tatgtggacc aaaaaaatct 600 actaatctgg tgaagaataa atgcgtcaat ttt taa 636 <210> 22 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> OVA257-264 Codon Optimized Construct DNA Sequence <400> 22 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 agtattagat t tttgagaa aa ct 120 cttggaggag gtggatcttc cataattaat tttgaaaagc tggattataa ggatgacgac 180 gataagtga 189 <210> 23 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> OVA323-339 Codon Optimized Construct DNA Sequence <400> 23 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 atttcacagg ctgtgcatgc tgcacatgct gaaattaatg aggctggacg tgattataag Optimized DNA Sequence Optimized Sequence 400 gatgacgacg ataagt 213 <138 <k> 24 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 gctagcatga cgaacatgga gctaatggga gggggcggaa gtgcttctat gactaatatg 120 gaactgatgg gaggtggagg ttctgcttca atgaccaaca tggaacttat ggattataag 180 gatgacgacg ataagtga 198 <210> 25 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> Rpl18 Codon Optimized Construct DNA Sequence <400> 25 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 aagattctga cgtttgatcg tctggggggt ggcggatcta agatattgac gttcgatcga 120 ctgggaggag gtggctctaa gatcctaaca ttcgaccgtc tagattataa ggatgacgac 180 gataagtga 189 <210> 26 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> P15E Codon Optimized Construct DNA Sequence <400> 26 gggaggtggag gctcaggt gg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 aagtcacctt ggtttactac tctcggtggg ggaggaagta aatcgccatg gtttactaca 120 ttgggaggag gtgggtctaa gtcaccatgg ttcacgacat tggattataa ggatgacgac 180 gataagtga 189 <210> 27 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> S21P2(1) Codon Optimized Construct DNA Sequence <400> 27 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 cctagtaagc cttctaagcg cagcttcata gaagatttgt tgttcaataa ggtagattat 210 Sequencing 220 aaggat <agtg < 212 Artificial> 28223 <agtgacg < acga (2) Codon Optimized Construct DNA Sequence <400> 28 ggaggtggag gctcaggtgg cggaggttct gagcagaagt tgatttcaga ggaagatctg 60 cctagtaagc cttctaagcg cagcttcata gaagatttgt tgttcaataa ggtaggtggg 120 ggaggaagtc catcgaagcc aagtaagcgt agtttcattg aggacctcct cttcaacaag 180 gttgattata aggatgacga cgataagtga 210 <210> 29 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> S1 fwd <400> 29 cacccaggga ctcgatgatg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> S1 rev <400> 30 gcacccaact gggtaacact 20 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> RBD fwd <400> 31 caactgagca aagttacgtg agggtacaac ctacggaatc 40 <210> 32 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> RBD rev <400> 32 atgaaatgcc ccagtgccgc ggggtggtct gacctcaaaa attgacgcat ttattcttca 60 c 61 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> S1-250-AvrII-F <400> 33 gattctatca actcaaggat aggcgcaatt gagcctagg 39 <210> 34 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> pBacT7-SacII-R<400> 34 cagtgccgcg gggtggtctg atcctcacgt gaaactacgc gggta 45

Claims (16)

the amino acid sequence of the attenuated reovirus sigma 1 protein; And in a polypeptide comprising an epitope amino acid sequence of an antigen causing cancer or an infectious disease,
amino acid sequence 1 to 250 of the reovirus sigma 1 protein attenuated by truncating at amino acid position 251 at the N-terminus; and
The attenuated reovirus sigma 1 protein is inserted into the amino acid position after 251 at the N-terminus of the amino acid sequence of the protein, characterized in that it contains the amino acid sequence of the epitope of an antigen causing cancer or infectious disease, characterized in that it comprises a polypeptide .
According to claim 1,
The attenuated reovirus sigma 1 protein is characterized in that represented by the amino acid sequence of SEQ ID NO: 1, a polypeptide.
3. The method of claim 2,
The attenuated reovirus sigma 1 protein is characterized in that encoded by the nucleotide sequence shown in SEQ ID NO: 2, a polypeptide.
According to claim 1,
wherein the epitope forms or constitutes a fusion protein at the carboxy terminus of the attenuated reovirus sigma 1 protein.
According to claim 1,
Before and after the amino acid sequence of the epitope, a linker (SEQ ID NO: 11), Myc protein (SEQ ID NO: 12), FLAG protein (SEQ ID NO: 13), and at least one amino acid sequence selected from the group consisting of 2A peptides further comprising with, a polypeptide.
According to claim 1,
The cancer is liver cancer, glioma, sarcoma, colorectal cancer, breast cancer, prostate cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer, stomach cancer, esophageal cancer, bile duct cancer, pancreatic cancer, cervical cancer, skin cancer, lymphoma, thyroid cancer, bone marrow cancer, The polypeptide, characterized in that at least one selected from the group consisting of endometrial cancer, kidney cancer, rectal cancer, and brain tumor.
According to claim 1,
The infectious disease is Corona Virus Infectious Disease-19 (COVID-19), Hepatitis C, Influenza, Human Immunodeficiency Virus (HIV)-induced AIDS, Tuberculosis, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) , Infantile enteritis caused by rotavirus, and non-bacterial acute gastroenteritis caused by norovirus, characterized in that at least one selected from the group consisting of, a polypeptide.
According to claim 1,
The amino acid sequence of the epitope is characterized in that at least one selected from the group consisting of SEQ ID NOs: 3 to 10, the polypeptide.
According to claim 1,
The nucleotide sequence encoding the epitope is characterized in that it is substituted or inserted at positions 763 to 1416 nucleotides from the 5' end of the nucleotide sequence encoding the attenuated reovirus sigma 1 protein, the polypeptide.
According to claim 1,
The epitope is characterized in that it causes an immune response by generating a neutralizing antibody or inducing cellular immunity in the host, the polypeptide.
According to claim 1,
The epitope is a polypeptide, characterized in that at least one selected from the group consisting of CD4+ T cells, CD8+ T cells, and B-cell epitopes.
An attenuated reovirus-based viral vector comprising a polynucleotide encoding the polypeptide of any one of claims 1 to 11.
An attenuated reovirus-based vaccine composition comprising effectively the polypeptide of any one of claims 1 to 11, a polynucleotide encoding the same, or the viral vector of claim 12.
14. The method of claim 13,
The vaccine composition is characterized in that for the prevention or treatment of cancer or infectious disease, the vaccine composition.
A method for preparing an attenuated reovirus-based vaccine composition comprising the steps of:
a) preparing a reverse-evolved viral vector to include a nucleotide sequence encoding the attenuated reovirus sigma 1 protein represented by SEQ ID NO: 1; and
b) At the 763 to 1416 nucleotide sequence positions of the nucleotide sequence encoding the sigma 1 protein, the nucleotide is substituted or inserted so as to include a nucleotide sequence encoding an epitope of an antigen causing cancer or infectious disease to be attenuated Introducing a polynucleotide encoding the reovirus sigma 1 protein and an epitope of an antigen causing cancer or an infectious disease to be expressed by fusion into the vector.
16. The method of claim 15,
c) producing and propagating the attenuated reovirus-based vector comprising the polynucleotide of step b) in one or more cells selected from the group consisting of BHK21, L929, HEK293, CHO, PER.C6, HeLa, and Vero cells A method for producing a vaccine composition, characterized in that it comprises the step of:

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